Constitutive exo-pectinase produced byAspergillus sp. CH-Y-1043 on different carbon source

Summary The production of a constitutive exo-pectinase byAspergillus sp. CH-Y-1043 grown on glucose, sucrose, fructose, glycerol and galacturonic acid is reported. The specific activity was found to be in the range of 26% to 75% of that produced with pectin or poly-galacturonic acid. The production of this exo-pectinase is strictly correlated to the exponential growth phase and it is highly sensitive to the pH of the culture medium     

A monoclonal antibody specific for conidia and mycelium wall layer of Penicillium and Aspergillus.

A monoclonal antibody was obtained from BALB/c mice immunized with Penicillium frequentans mycelium. The specificity of the antibody was evaluated by enzyme-linked immunosorbent and indirect immunofluorescence assays against the same mycelium. This IgM antibody cross-reacted with various strains of the Penicillium and Aspergillus genera. By indirect immunofluorescence assays, the antibody was able to stain about 10% of Penicillium and Aspergillus conidia, but major part of conidia did not absorb the fluorescence-labeled antibody before swelling. During germination of P. frequentans conidia, the germ tube wall which constitutes a continuation of an inner wall layer was also stained. During germination of P. griseofulvum, the protrusion of the germ tube wall was not always recognized by the antibody because the germ tube wall was constituted by a continuation of an outer spore wall layer. The study of the staining patterns of the spores and the protrusions suggests that the antibody specifically recognizes an antigen of the inner spore wall layer. The monoclonal antibody reacts with extracellular galactomannans produced by genera Aspergillus and Penicillium but is not directed against beta-(1,5)-linked galactofuranose units.     

Release of proteinase from mycelium of Mucor hiemalis

When Mucor hiemalis NRRL 3103 was grown in soybean medium, only a small fraction of the proteinase produced by the organism appeared in the culture filtrate, whereas the bulk of the enzyme was bound to the mycelial surface. Optimal pH of the proteinase ranged from 3.0 to 3.5. Inclusion of sodium chloride or other ionizable salts in the growth medium, however, resulted in the liberation from the mycelium of the loosely bound enzyme as it was formed. Maximal release of proteinase was achieved at a sodium chloride concentration of 0.5 m. The loosely bound proteinase was eluted also from intact resting mycelium by ionizable salts but not by water or by nonionizable substances. The amount of enzyme eluted from the mycelium depended upon the concentration of sodium chloride up to 0.3 m. Since liberation took place rapidly even at 0 C, a loose ionic linkage must exist rather than a biochemical binding of the enzyme to the mycelium. The recovery of proteolytic activity from repeated salt extractions was greater than that originally detected in the intact mycelium, possibly owing to unmasking of more active enzymes or functional groups. Further proteinase activity was released when salt-extracted mycelium was ruptured. Part of the proteinase thus observed was firmly attached to the cell fraction, and part of it appeared in the supernatant fluid. These conditions implied the presence of intracellular or firmly attached proteinase which could be partially released.     

Protoplasts from Aspergillus nidulans.

A very effective lytic enzyme system for massive micro/macro-scale production of protoplasts from the filamentous fungus Aspergillus nidulans is described. A striking coincidence was observed between maximal lytic activity towards Aspergillus mycelium and the presece of both chitinase and alpha-(1 leads to 3)-glucanase activities. The release of protoplasts was greatly enhanced by preincubating the mycelium with 2-deoxy-D-glucose. Furthermore, protoplast formation was influenced by fungal age, culture conditions, pH of incubation and the osmotic stabilizer used. From 40 mg of fresh mycelium, grown for 14--16 h on 1% glucose in a low phosphate-citrate medium, preincubated with 2-deoxy-D-glucose for 45 min, and then incubated with the lytic enzyme mixture at pH 6.5 in the presence of 0.3--0.4 M (NH4) SO4, 2.5 x 10(8) stable protoplasts were produced within 3 h of incubation at 30 degrees C. Comparable results were obtained with 40--50 g of mycelium. At low osmotic stabilizer concentrations a peculiar type of regeneration was observed in the presence of the lytic enzyme system; within 12 h of incubation aberrant hyphal structure emerged from the large vacuolated protoplasts.     

Changes in development and ultrastructure of Aspergillus niger mycelium in the presence of toxic substances from molasses in citric acid fermentation medium.

Effects of a surface-active agent (Spumol BJ) and toxic molasses compounds on development and ultrastructure of Aspergillus niger strain R-16 mycelium producing citric acid in surface fermentation on molasses medium with 80% yield are presented. Microscopic observations showed that Spumol BJ in concentration of 5 microliters/100 cm3 as well as toxic molasses compounds stimulated the process of swelling and germinating of conidia. Giant conidia unable to germinate, appeared along with typical ones. Delicate mycelium outgrowing from germinating conidia however, sank after 20-24 h culture. Observations of hyphae in electron microscope showed changes in the ultrastructure and dimensions of mitochondria followed by successive degeneration of protoplast. The data obtained indicate that one of the reasons disqualifying raw molasses for biotechnological processes might be the excessive amount of surface-active antifoaming additives.     

Effect ofAspergillus parasiticus soil inoculum on invasion of peanut seeds

Environmental control plots adjusted to late season drought and elevated soil temperatures where inoculated at peanut planting with low and high levels of conidia, sclerotia, and mycelium from a brown conidial mutant ofAspergillus parasiticus. Percentage infection of peanut seeds from undamaged pods was greatest for the subplot containing the high sclerotial inoculum (15/cm² soil surface). Sclerotia did not germinate sporogenically and may have invaded seeds through mycelium. In contrast, the mycelial inoculum (colonized peanut seed particles) released large numbers of conidia into soil. Soil conidial populations of brownA. parasiticus from treatments with conidia and mycelium were positively correlated with the incidence of seed infection in undamaged pods. The ratio ofA. flavus to wild-typeA. parasiticus in soil shifted from 7:3 to 1:1 in the uninoculated subplot after instigation of drought, whereas in all subplots treated with brownA. parasiticus, the ratio of the two species became approximately 8:2. Despite high levels of brownA. parasiticus populations in soil, nativeA. flavus often dominated peanut seeds, suggesting that it is a more aggressive species. Sclerotia of wild-typeA. parasiticus formed infrequently on preharvest peanut seeds from insect-damaged pods.     

Improvement of pectinase production by interspecific hybrids of Aspergillus strains

Protoplast fusion induced by polyethylene glycol and Ca²⁺, was performed between auxotrophic mutants of pectinolytic fungi Aspergillus sp. CH-Y-1043 (A13) ade ⁻ and Aspergillus flavipes ATCC-16795 (F7) lys ⁻. Prototrophic colonies were developed on minimal medium with a fusion frequency of 1·0×10⁻². The reversion frequency of the mutation in spores and protoplasts was low and ranged from 2·0 to 4·0×10⁻⁷. Four prototrophic hybrids (HH, HE, HF and HJ) exhibited enhanced production of endo-pectinase and pectin-lyase. The highest production was observed in HJ ; maximum activities were 150 and 160% respectively, whereas the exo-pectinase production was similar to the wild-type strain Aspergillus sp. CH-Y-1043. Hybrid HJ showed the greatest growth ; nevertheless, specific endo-pectinase and pectin-lyase activities were higher in all hybrids than those produced by the wild-type strains.     

Partitioning of ochratoxin A in mycelium and conidia of Aspergillus carbonarius and the impact on toxin contamination of grapes and wine

AIMS: Aspergillus carbonarius is an important ochratoxin A (OTA)-producing fungus which is responsible for toxin contamination of grapes and wine. The objectives of this study were to examine the partitioning of OTA in mycelium and conidia of a range of A. carbonarius strains on artificial grape juice and defined media, to determine the excretion patterns of OTA from these spores, and the effect of organic acids used in wine production on OTA excretion from conidia. METHODS AND RESULTS: The results showed that 60-70% of the OTA was accumulated in the conidia of a number of different isolates of A. carbonarius. Calculations showed that on different defined media, an amount of 0.011- to 0.1-pg OTA was present per conidium. The OTA in spores was found to be rapidly excreted into the medium during the initial few hours after conidial germination leading to an increase of OTA in must during maceration for wine production. The presence of tartaric acid inhibited OTA production, but malic acid enhanced this production during mycelial growth. These acids were also shown to affect the time course of germination and the rate of OTA excretion from conidia during germination. CONCLUSIONS: This study is the first to examine and show the partitioning of OTA into spores of strains of A. carbonarius and that rapid excretion of OTA from spores could be a reason for OTA accumulation in musts during wine production. SIGNIFICANCE AND IMPACT OF THE STUDY: Conidia of A. carbonarius could be a major source of OTA contamination of grapes used in wine production. This information could help in the development of effective prevention strategies to minimize wine contamination with this important mycotoxin.     

Production of pectinase from deseeded sunflower head by Aspergillus niger in submerged and solid-state conditions

Studies were carried out on the production of pectinases using deseeded sunflower head by Aspergillus niger DMF 27 and DMF 45 in submerged fermentation (SmF) and solid-state fermentation (SSF). Higher titres of endo- and exo-pectinases were observed when medium was supplemented with carbon (4% glucose for SmF and 6% sucrose for SSF) and nitrogen (ammonium sulphate, 0.3% for both SmF and SSF) sources. Green gram husk proved to be relatively a better supplement to attain higher yield of endo-pectinase (11.7 U/g) and exo-pectinase (30.0 U/g) in solid-state conditions. Maximum production of endo-pectinase (19.8 U/g) and exo-pectinase (45.9 U/g) by DMF 45 were recorded in SSF when compared to endo-pectinase (18.9 U/ml) and exo-pectinase (30.3 U/ml) by DMF 27 in SmF under optimum process conditions.     

Influence of a pulsed electric field on the spores and oxygen consumption of Aspergillus niger and its citric acid production

Spores of Aspergillus niger were exposed to a pulsed electric field. After treatment by the electric field, the viability of the conidia of A. niger varied depending on the field strength, pulse width and frequency. In all cases, these parameters reduced the viability rate of the conidia from 2.0 × 10⁷ to a range from 6.2 × 10⁶ to 8.5 × 10⁶ spores/ml (3.1 to 42.6%). After pulse treatment, the conidia were used as the inoculum for citric acid fermentation in shake flasks. The highest increase in citric acid yield (about 1.4-fold) was reached at a field strength of 2.85 kV/cm, a frequency of 1 Hz and a pulse width of 1 ms. When the parameters of the electric field increased there were important changes in the respiration rate of the Aspergillus niger mycelium (48-h-old) after electric shock treatment. The highest consumption of dissolved oxygen (22.9%) in the medium by Aspergillus niger mycelium was observed at an electric field strength of 2.85 kV/cm, a 1 Hz frequency, a pulse width of 1 ms and a 1-min exposure period. It seems that an electric-field stimulation of the conidia prior to inoculation may offer an important method of improving the efficiency of citric acid. The treatment of the conidia is both simple from the technical point of view and extremely rapid.     

Morphology engineering of Aspergillus niger for improved enzyme production

Supplementation with silicate microparticles was used as novel approach to control the morphological development of Aspergillus niger, important as the major world source of citric acid and higher‐value enzymes, in submerged culture. With careful variation of size and concentration of the micromaterial added, a number of distinct morphological forms including pellets of different size, free dispersed mycelium, and short hyphae fragments could be reproducibly created. Aluminum oxide particles similarly affected morphology, showing that this effect is largely independent of the chemical particle composition. Image analysis of morphological development of A. niger during the cultivation process showed that the microparticles influence the morphology by collision‐induced disruption of conidia aggregates and probably also the hindrance of new spore–spore interactions in the very early stage of the process. Exemplified for different recombinant A. niger strains enzyme production could be strongly enhanced by the addition of microparticles. Linked to the formation of freely dispersed mycelium, titers for glucoamylase (GA) expressed as intracellular enzyme (88 U/mL) and fructofuranosidase secreted into the supernatant (77 U/mL), were up to fourfold higher in shake flasks. Moreover, accumulation of the undesired by‐product oxalate was suppressed by up to 90%. The microparticle strategy could be successfully transferred to fructofuranosidase production in bioreactor, where a final titer of 160 U/mL could be reached. Using co‐expression of GA with green fluorescent protein, enzyme production was localized in the cellular aggregates of A. niger. For pelleted growth, protein production was maximal only within a thin layer at the pellet surface and markedly decreased in the pellet interior, whereas the interaction with the microparticles created a highly active biocatalyst with the dominant fraction of cells contributing to production. Biotechnol. Bioeng. 2010;105: 1058–1068. © 2009 Wiley Periodicals, Inc.     

Lipase of <Emphasis Type="Italic">Aspergillus niger</Emphasis> NCIM 1207: A Potential Biocatalyst for Synthesis of Isoamyl Acetate

Commercial lipase preparations and mycelium bound lipase from Aspergillus niger NCIM 1207 were used for esterification of acetic acid with isoamyl alcohol to obtain isoamyl acetate. The esterification reaction was carried out at 30°C in n-hexane with shaking at 120 rpm. Initial reaction rates, conversion efficiency and isoamyl acetate concentration obtained using Novozyme 435 were the highest. Mycelium bound lipase of A. niger NCIM 1207 produced maximal isoamyl acetate formation at an alcohol/acid ratio of 1.6. Acetic acid at higher concentrations than required for the critical alcohol/acid ratio lower than 1.3 and higher than 1.6 resulted in decreased yields of isoamyl acetate probably owing to lowering of micro-aqueous environmental pH around the enzyme leading to inhibition of enzyme activity. Mycelium bound A. niger lipase produced 80 g/l of isoamyl acetate within 96 h even though extremely less amount of enzyme activity was used for esterification. The presence of sodium sulphate during esterification reaction at higher substrate concentration resulted in increased conversion efficiency when we used mycelium bound enzyme preparations of A. niger NCIM 1207. This could be due to removal of excess water released during esterification reaction by sodium sulphate. High ester concentration (286.5 g/l) and conversion (73.5%) were obtained within 24 h using Novozyme 435 under these conditions.     

Virus Particles from Conidia of Penicillium Species

Virus particles and their component double-stranded ribonucleic acid (dsRNA) have been isolated from conidia and mycelia of certain Penicillium species. The conidia and mycelia of P. stoloniferum NRRL 5267 contained 75 and 85 mug of dsRNA/g (dry weight), respectively. Of the total dsRNA released from NRRL 5267 conidia, 10% was nonencapsulated. Conidia of P. brevi-compactum NRRL 5260 and P. chrysogenum Q-176 contained 2 and 120 mug of dsRNA/g (dry weight), respectively, whereas mycelium from the two species contained 3 and 95 mug of dsRNA/g (dry weight), respectively. No viruses were isolated from conidia or mycelia of P. stoloniferum NRRL 859. A method is described for disruption of both conidia and mycelia. The technique facilitates the isolation and characterization of fungal viruses and their component dsRNA and also potentiates surveying of fungal isolates for the presence of virus.     

Production of acid proteinases by Humicola lutea mycelium immobilized in polyurethane sponge.

Humicola lutea 120-5 spores were entrapped in polyurethane sponge cubes and were cultivated inside the carrier to form an immobilized mycelium further used for production of acid proteinases in batch mode. A carrier—spore suspension ratio of 10:0.5 (wt) should be used to obtain optimal results. The polyurethane sponge-immobilized mycelium could be applied repeatedly, the enzyme activity secreted during the first 10 cycles being about the same as that produced by free cells. The advantages of immobilizing fungal cells by germinating conidia entrapped inside the supporting material are discussed.     

The preparation and chemical composition of fractions from Aspergillus fumigatus wall and protoplasts possessing antigenic activity.

A detergent-soluble fraction was prepared from the fragmented wall of Aspergillus fumigatus mycelium using the non-ionic detergent Triton X-100, and a wall-free extract was prepared from the same source in the form of protoplasts, released by a lytic enzyme system from Trichoderma harzianum. These extracts were examined by polyacrylamide gel electrophoresis and their detailed chemical composition was established. They were compared with the water-soluble fraction prepared from total mycelium, which is used routinely in this laboratory for serological tests. All fractions had immunological reactivity towards an antiserum prepared in rabbits against this water-soluble fraction of the mycelium, as shown by double diffusion. Both protein and carbohydrate moieties appear to be involved in the antigenic sites, with carbohydrate reactivity predominantly associated with the protoplast fraction. The fact that all preparations contained at least one common antigenic determinant, as judged by lines of identity to a single antiserum, is discussed in relation to antigen location.     

Characteristics of exogenous dormancy of Aspergillus niger conidia]

Aspergillus niger conidia are characterized by exogenous dormancy: the first stage of their germination is accomplished in twice distilled water. However, germ tube formation requires the availability of carbon and nitrogen sources. Exogenous dormancy in A. niger conidia exhibits the following peculiar features: (i) nitrogen-containing substances are active stimulators of germination; (ii) temperature-dependent changes in the lipid bilayer and in the neutral lipid composition of conidia are virtually identical to those occurring in growing mycelium under temperature stress; and (iii) the spore viability threshold does not exceed 45 degrees C; i.e., the spores are more heat-resistant than the mycelium, but they are less heat-resistant than the spores that are in the state of endogenous dormancy. According to the current classification of the types of cell metabolism arrest, the exogenous dormancy of A. niger conidia resembles the pattern of metabolism characteristic of vegetative cells during the idiophase.     

Glycosylphosphatidylinositol-anchored Ecm33p influences conidial cell wall biosynthesis in Aspergillus fumigatus

ECM33 encodes a glycosylphosphatidylinositol-anchored protein whose orthologs in yeast are essential for sporulation. Aspergillus fumigatus Ecm33p is unique and has an apparent mass of 55 kDa. Disruption of A. fumigatus ECM33 results in a mutant with several morphogenetic aberrations, including the following: (i) a defect in conidial separation, (ii) an increase in the diameter of the conidia of the mutant associated with an increase in the concentration of the cell wall chitin, (iii) conidia that were sensitive to the absence of aeration during long-term storage, and (iv) conidia that were more resistant to killing by phagocytes, whereas the mycelium was more easily killed by neutrophils.     

Gene cloning and functional characterization by heterologous expression of the fructosyltransferase of Aspergillus sydowi IAM 2544

We have purified a fructosyltransferase from conidia of the inulin-producing fungus Aspergillus sydowi IAM 2544 and obtained peptide sequences from proteolytic fragments of the protein. With degenerated primers, we amplified a PCR fragment that was used to screen a cDNA library. The fructosyltransferase gene from Aspergillus sydowi (EMBL accession no. AJ289046) is expressed in conidia, while no expression could be detected in mycelia by Northern blot analysis of mycelial RNA. The gene encodes a protein with a calculated molecular mass of 75 kDa that is different from all fructosyltransferases in the databases. The only homology that could be detected was to the invertase of Aspergillus niger (EMBL accession no. L06844). The gene was functionally expressed in Escherichia coli, yeast, and potato plants. With protein extracts from transgenic bacteria and yeast, fructooligosaccharides could be produced in vitro. In transgenic potato plants, inulin molecules of up to 40 hexose units were synthesized in vivo. While in vitro experiments with protein extracts from conidia of Aspergillus sydowi yielded the same pattern of oligosaccharides as extracts from transformed bacteria and yeast, in vivo inulin synthesis with fungal conidia leads to the production of a high-molecular-weight polymer.     

Peculiarities of Exogenous Dormancy of Aspergillus nigerConidia

Aspergillus nigerconidia are characterized by exogenous dormancy: the first stage of their germination is accomplished in twice-distilled water. However, germ tube formation requires the availability of carbon and nitrogen sources. Exogenous dormancy in A. nigerconidia exhibits the following peculiar features: (i) nitrogen-containing substances are active stimulators of germination; (ii) temperature-dependent changes in the lipid bilayer and in the neutral lipid composition of conidia are virtually identical to those occurring in growing mycelium under temperature stress; and (iii) the spore viability threshold does not exceed 45°C; i.e., the spores are more heat-resistant than the mycelium, but they are less heat-resistant than the spores that are in the state of endogenous dormancy. According to the current classification of the types of cell metabolism arrest, the exogenous dormancy of A. nigerconidia resembles the pattern of metabolism characteristic of vegetative cells during the idiophase.     

Seeds as natural matrices for immobilization of Aspergillus niger mycelium producing pectinases

J. FIEDUREK, J. SZCZODRAK AND J. ROGALSKI. 1995. A simple method for the immobilization of Aspergillus niger mycelium producing polygalacturonase (PG) and pectinesterase (PE) is described. Fungal conidia were immobilized on wheat, rye, barley, peas, buckwheat and mustards seeds. Spongy mycelia overgrowing the seed surfaces on mineral medium with pectin produced extracellular PG and PE; the highest production was reached on the wheat carrier. Some of the variables influencing the enzymatic activity have been optimized. After every 24 h, a culture liquid with 6˙8–7˙8 U of PG ml⁻¹ and 7˙0–10˙1 U of PE ml⁻¹ was obtained. This procedure also made possible repeated batch enzyme production and, as many as eight subsequent 24-h batches could be fermented by using the same carrier without any loss of PG activity. The addition of sodium orthovanadate (1 mmol) into the medium with pectin caused a significant increase in PG and PE activity produced by free cells of A. niger (by 1˙59-fold and 1˙67-fold respectively), and only 0˙47-fold of PG activity in case of the immobilized mycelium.